Process for the production of (−)-epigallocatechin gallate

ABSTRACT

A process is provided for making (−)-epigallocatechin gallate (EGCG) by subjecting a green tea extract to chromatography on a macroporous polar resin, eluting EGCG from the resin with a polar elution solvent, optionally concentrating the eluate, optionally regenerating the resin by desorbing the remaining catechins, and optionally concentrating the desorbed catechins.

This is a Continuation of U.S. application Ser. No. 09/638,926, filedAug. 16, 2000 abandoned.

FIELD OF THE INVENTION

The present invention relates to a process for the production of(−)-epigallocatechin gallate (EGCG). The invention particularly relatesto a process for the production of EGCG by separation from tea catechinsby treatment with a macroporous polar resin.

BACKGROUND OF THE INVENTION

Leaves of the green tea plant camellia sinensis contain up to 36%polyphenols on a dry weight basis, however, their composition varieswith climate, season, variety and state of maturity. Green tea catechinsare the predominant group of green tea polyphenols. Examples ofcatechins are (−)-epicatechin (EC), (−)-epigallocatechin gallate (EGCG),epigallocatechin (EGC) and epicatechin gallate (ECG).

EGCG is the most interesting compound among the above mentionedcatechins because it exhibits a strong antioxidant effect. Furthermore,it has been demonstrated that EGCG has an antimutagenic effect, anantibacterial effect, and a beneficial effect on cholesterol level inblood. The other catechins present in green tea are much less effectivecompared to EGCG. Green tea also contains other components such ascaffeine, proteins, pectins, and/or metal ions which might not bedesirable.

There is, therefore, a need to isolate EGCG in a pure form in high yieldby a simple and economical process. However, the structural similaritiesof the various green tea catechins make the separation of the individualcatechins difficult. Furthermore, the catechins in green tea arenormally accompanied by caffeine, which is present in an amount up to 4%of the dry mass of the green tea leaves. Caffeine is known to associatewith the catechins and is not trivial to remove.

The production of green tea extracts is well known in the art.Ekanayake, et al., U.S. Pat. No. 5,879,733 (“Ekanayake '733”) describesthe preparation of a green tea extract having improved clarity andcolor. The green tea extract is obtained by treating the extract at atemperature in the range of 25° C. to 60° C. with an amount of a foodgrade cation exchange resin effective to remove metal cations present inthe extract. The treated extract is then contacted with a nanofiltrationmembrane. However, the process described in Ekanayake '733 is notsuitable to separate EGCG from a mixture of tea catechins.

Hara, U.S. Pat. No. 4,613,672 (“Hara '672”) describes a process for thepreparation of pure EGCG which process includes the following steps: Tealeaves are extracted with hot water or with aqueous solutions of 40-75%methanol, 40-75% ethanol or 30-80% acetone. The obtained extract iswashed with chloroform, and the washed extract is dissolved in anorganic solvent. The organic solvent is distilled off, and theconcentrated extract component is subjected to high speed liquidchromatography using a reverse-phase partition column with a developerof acetone/tetrahydrofuran/water (0-25:0-35:65-85, vol %), whereby eachof (−) epicatechin, (−) epigallocatechin, (−) epicatechin gallate and(−) epigallocatechin gallate is isolated from one another. The processdescribed in Hara '672 does not permit an economical production of EGCGon a technical scale because of the use of expensive column fillings.Furthermore, the process described in Hara '672 does not permit theproduction of EGCG, which may be added to food products because thesolvent mixture used (i.e., acetone/tetrahydrofuran/chloroform) is notfood-approved.

SUMMARY OF THE INVENTION

While the art describes the production of catechins as mixtures, thereis still a need for a simple, safe, and economic process for producingEGCG in a purified form for incorporation as an ingredient intosupplements and foodstuffs.

One object of the present invention is to provide a process forproducing EGCG in a purified form for incorporation as an ingredientinto supplements and foodstuffs.

Another object of the invention is a process to separate EGCG from amixture of tea catechins and/or caffeine with improved selectivity whencarrying out the separation using a macroporous polar resin and asuitable polar elution solvent.

Accordingly, one embodiment of the invention is a process for producingepigallocatechin gallate (EGCG) comprising the steps of:

-   -   a) providing a green tea extract;    -   b) subjecting the green tea extract to chromatography on a        macroporous polar resin at a temperature between about 10° C. to        about 80° C.; and    -   c) eluting the EGCG from the macroporous polar resin with a        polar elution solvent at a temperature between about 10° C. to        about 80° C. and at a pressure between about 0.1 bar to about 50        bar.

DETAILED DESCRIPTION OF THE INVENTION

Thus, the present invention is directed to a process for producingepigallocatechin gallate (EGCG), which includes the steps of:

a) providing a green tea extract;

b) subjecting the green tea extract to chromatography on a macroporouspolar resin at a temperature in the range of about 10° C. to about 80°C.;

c) eluting EGCG from the macroporous polar resin with a polar elutionsolvent at a temperature in the range of about 10° C. to about 80° C.and at a pressure in the range of about 0.1 bar to about 50 bar;

d) optionally concentrating the eluate of step c);

e) optionally regenerating the macroporous polar resin by desorbing theremaining catechins; and

f) optionally concentrating the desorbed catechins of step e).

The production of the green tea extract used as starting material iswell known in the art. For example, green tea leaves are typicallyextracted with hot or cold water to form a solution containing teacatechins and caffeine. This green tea solution may be furtherconcentrated to form either a concentrated extract solution or a drypowder. The extract solution or the powder may contain stabilizers, suchas food-approved acids, e.g. citric acid, ascorbic acid, isoascorbicacid, and the like.

Tea extract powders, such as green tea extract powders, are alsocommercially available from, for example, Guizhou Highyin BiologicalProduct Co., Guiyang, P.R. China, or Zhejang Zhongke Plant Technical Co.Ltd., Hangzhou, Zhejang, P.R. China.

The separation of EGCG may be carried out by subjecting the green teaextract to a column filled with a macroporous polar resin.

As used herein, “macroporous polar resins” means acrylic resins, such aspolyacrylates, e.g. AMBERLITE® XAD-7 (available from Rohm and Haas,Philadelphia, Pa.); polymethacrylates (such as e.g. AMBERCHROM® CG-71available from Toso Haas or DIAION HP 2MG available from MitsubishiChem. Corp., Philadelphia, Pa.); polyamides (such as Polyamide 11available from Merck, Darmstadt, Germany, Polyamide 6 and Nylon 6,6available from Fluka, Buchs, Switzerland catalogue Nos. 02395 and 74712,respectively, and Polyamide 12, Grilamid L 25 natur, available from EMSChemie, Domat, Switzerland); Polyvinylpyrrolidone P 6755 (available fromSigma); aromatic polyamides; and polyesters.

The resin is preferably operated degassed and equilibrated with theelution solvent.

The process according to the invention is performed at temperatures inthe range of about 10° C. to about 80° C., preferably of about 40° C. toabout 60° C. Thermostatic control may take place, for example, byplacing the column in a thermostatically controlled area, such as, aheating jacket.

The hydraulic pressure under which the mobile phase is passed throughthe column may be varied within wide limits. The mobile phase ispreferably pumped through the column at a pressure of about 0.1 bar toabout 50 bar, preferably at about 0.1 bar to about 20 bar, such as, forexample, at about 0.1 bar to about 10 bar.

The mobile phase includes a polar elution solvent which is a mixture ofwater and an organic solvent. As used herein, “organic solvent” meansalcohols, such as methanol, ethanol, isopropanol and the like, andketones, such as acetone or esters such as ethylacetate or mixturesthereof. The use of food grade alcohols, such as ethanol andisopropanol, is preferred. Particularly good results are obtained whenusing a mobile phase containing a mixture of about 70 vol % to about 95vol %, preferably about 90 vol %, of water and about 5 vol % to about 30vol %, preferably about 10 vol %, of organic solvent. It is advantageousto degas the mobile phase and keep it under an inert atmosphere, such asnitrogen or argon.

The column is conditioned with the mobile phase. The flow rate of themobile phase through the column may be varied within wide limits. Theflow rate is in the range of about 0.5 to about 20 bed volumes/hour,preferably about 0.5 to about 10 bed volumes/hour, such as about 0.8 toabout 5 bed volumes/hour. (1 bed volume corresponds to 1 m³ solution orsolvent per m³ resin).

After equilibrium has been established between the stationary and mobilephases, tea extract solution is introduced into the mobile phase, thus,subjecting the green tea extract to chromatography on the macroporouspolar resin. If a green tea extract powder is used as the startingmaterial, the powder is dissolved in the mobile phase. If an aqueousgreen tea extract is used, it is advantageous to adjust the ratio ofwater to organic solvent in the extract to that of the mobile phase byadding an organic solvent.

A key aspect of the present invention is to treat the green tea extractwith a macroporous polar resin at temperatures in the range of about 10°C. to about 80° C., preferably at about 40° C. to about 60° C. and toelute EGCG with a polar elution solvent. This particular interplay ofthe three features of resin, eluent, and temperature forms an importantaspect of the present invention and leads to a specific separation ofEGCG from a mixture of tea catechins and/or caffeine, thus obtaining,after elution, an EGCG fraction containing at least 75%, preferably morethan 85%, such as for example, about 90% to about 97%, of EGCGcalculated based on the total amount of catechins present in the extractor concentrate.

The ability of the macroporous polar resin to absorb caffeine, EGCG andthe remaining catechins is different depending also on the eluent usedand the temperature. The affinity of the resin for caffeine is less thanthat for EGCG, thus, caffeine, if present, is eluted first and may beseparated off. If appropriate, it is also possible to recover caffeinein a purified form using this process, which could be an economicadvantage, too. A second fraction is isolated in which the EGCG ispresent. The remaining tea catechins show a stronger affinity for theresin than EGCG does, thus, remaining adsorbed until the resin isregenerated using a solvent which is able to desorb the remainingcatechins. For example, the remaining catechins may be desorbed byeluting with a pure organic solvent or by changing the ratio of water toorganic solvent in the mobile phase. A suitable regeneration solvent is,e.g., a pure organic solvent or a mixture of about 10 vol % to about 60vol % of water and of about 40 vol % to about 90 vol % of organicsolvent, preferably about 40 vol % of water and about 60 vol % oforganic solvent.

The concentration of the EGCG in the eluate may be carried out bymethods well known in the art, such as for example by evaporation. TheEGCG eluate may be evaporated to dryness to form a powder containingEGCG in high purity or concentrated to allow crystallization. Theconcentration may be carried out by adding a stabilizer to the eluatesuch as a food-approved acid, e.g. citric acid, ascorbic acid,isoascorbic acid, and the like. The acid is preferably added in anamount of about 0.1 to about 2.5 vol % with respect to EGCG.

The pre-fraction containing the caffeine and the fraction of step e)containing the remaining catechins may be concentrated as describedabove.

The process may be carried out using a single column or a system ofmultiple chromatographic columns. The process may also be carried outusing “simulated moving bed chromatography” or “annular chromatography,”both of which are well known in the art.

The process of the present invention may be conducted with simple andeconomical operations, and is thus applicable to a large scaleproduction in respect of yield and handling.

EGCG prepared as described above possesses a strong antioxidant activityand may be used as an antioxidant for various foodstuffs, cosmetics,oils, and the like. In addition, EGCG has an antimutagenic effect, anantibacterial effect, and a beneficial effect on cholesterol level inblood. Thus, concentrates or pure EGCG are useful in health carepreparations.

The following example are provided to further illustrate the process ofthe present invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1 Separation of EGCG

A green tea extract containing the different catechins and caffeine(manufactured by Guizhou Highyin Biological Products Co., Guiyang, Chinaas “Green Tea Extract, min. 95% of polyphenols”) was used as startingmaterial. The concentration of the components in the green tea extractwere determined by HPLC using UV absorbance and expressed as wt.-%. Thecontent of EGCG, caffeine, other, catechins, as well as gallic acid inthe starting material is shown in Table 1.

TABLE 1 Concentration of the tea components in the starting material Teaextract Tea extract Example 1 Example 1 Relative Compound HPLC/wt.-%Percentage/% Gallic acid 0.01 0.0 Catechin 2.3 3.2 Caffeine 11.0 15.1EGCG 38.1 52.3 Epicatechin 5.2 7.11 GCG 6.6 9.1 ECG 9.7 13.3 Total 72.9100

33.5 1 (26 kg) of AMBERLITE® XAD-7 resin having a particle size of 0.3to 1.2 mm were filled into a pilot scale column having an inner diameterof 150 mm, a length of 2 m and a volume of 35.4 l. The column wasequipped with a heating jacket. The resin was thoroughly washed withwater and equilibrated with a mixture of water/isopropanol (ratio 9:1 byvolume). The apparatus and the solvents used were degassed and keptunder an inert nitrogen atmosphere prior to use.

The temperature of the filled column was maintained at 60° C. 0.4 kg ofthe above green tea extract (Table 1) containing 152.5 g of pure EGCGwere dissolved in 1.8 kg of a mixture of water/isopropanol (ratio 9:1 byvolume) and applied to the top of the column. EGCG was eluted from thecolumn by means of a pump under a pressure of 0.5 bar and at atemperature of 60° C. with a mixture of water/isopropanol (ratio 9:1 byvolume) at a constant flow rate of 50 kg/hour. After an initial eluateof 144 kg (prefraction), a main eluate of 174 kg was collectedcontaining 112 g of EGCG as the main polyphenol component. The EGCGconcentration in the main eluate was 0.064 wt %. The yield of separatedEGCG starting from 152.5 g EGCG in the tea extract was 73.5 wt %.

To regenerate the resin, the remaining catechins were desorbed byeluting with 78.3 kg of a mixture of water/isopropanol (ratio 4:6 byvolume). Before the next separation, the column was conditioned with 86kg of a mixture of water/isopropanol (ratio 9:1 by volume) in backwashmode at a flow rate of 120 kg/hour.

Table 2 illustrates the separation effect as shown by the relativepercentage of EGCG. The concentration of the tea components in the maineluate was determined by HPLC using UV absorbance and expressed as wt.-%or ppm.

TABLE 2 Concentration of the tea components in the main eluate Mainfraction Example 1 Rel. Compound HPLC/ppm Percentage/% Gallic acid 0 0.0Catechin 21 3.1 Caffeine 1 0.1 EGCG 644 92.1 Epicatechin 29 4.1 GCG 30.4 ECG 1 0.1 Total 699 100

Example 2

Example 1 was repeated using another lot of the “Green tea extract, min.95% of polyphenols” from Guizhou Highyin Biological Products Co. whichcontained the components shown in Table 3.

TABLE 3 Concentration of the tea components in the starting material Teaextract Tea extract of Example 2 Example 2 Compound HPLC/wt.-% Rel.Percentage/% Gallic acid 0.1 0.1 Catechin 1.4 1.9 Caffeine 13.8 18.8EGCG 35.1 47.9 Epicatechin 3.3 4.5 GCG 8.2 11.2 ECG 11.4 15.6 Total 73.2100

The washed column of Example 1 was maintained at 60° C., and used tocarry out the separation. 0.4 kg of the above green tea extract (Table3) containing 140.5 g of pure EGCG were dissolved in 1.8 kg of a mixtureof water/isopropanol (ratio 9:1 by volume) and applied to the top of thecolumn. The column was then eluted as described in Example 1. After aninitial eluate of 200 kg (prefraction), a main eluate of 117 kg wascollected containing 72.8 g of EGCG as the main polyphenol component.The EGCG concentration in the main eluate was 0.062 wt %. The yield ofseparated EGCG starting from 140.5 g of EGCG in the tea extract was 51.8wt %.

To regenerate the resin, the remaining catechins were desorbed byeluting with 100 kg of a mixture of water/isopropanol (ratio 4:6 byvolume). Before the next separation step, the column was conditionedwith 100 kg of a mixture of water/isopropanol (ratio 9:1 by volume) inbackwash mode at a flow rate of 120 kg/hour.

Table 4 illustrates the separation effect as shown by the relativepercentage of EGCG in the main eluate. The concentration of the teacomponents in the main eluate was determined by HPLC using UV absorbanceand expressed as ppm. Compared to Example 1, EGCG was obtained in ahigher percentage.

TABLE 4 Concentration of the tea components in the main eluate Mainfraction Example 2 Rel. Compound HPLC/ppm Percentage/% Gallic acid 0 0.0Catechin 10 1.6 Caffeine 1 0.2 EGCG 622 96.4 Epicatechin 2 0.3 GCG 6 0.9ECG 4 0.6 Sum 645 100

Example 3 Concentration of the Eluate

9008 kg of the eluate of the adsorption/desorption column prepared byrepeated runs as described in Example 2 were stabilized by addition of2% citric acid, calculated on the EGCG amount. The eluate wasconcentrated at a temperature of 40° C. and a pressure of 55 mbar usinga falling film evaporator made from stainless steel with a heat exchangesurface area of 1.1 m². The amount of catechins and caffeine of the feedsolution subjected to the evaporation unit is given in Table 5.

TABLE 5 Concentration of the tea components in the purified EGCGsolution subjected to evaporation Feed Falling film evaporator CompoundHPLC/ppm Rel. Percentage/% Gallic acid 0 0.0 Catechin 10 1.4 Caffeine 10.1 EGCG 712 96.2 Epicatechin 7 0.9 GCG 7 0.9 ECG 3 0.4 Total 740 100

The feed flow to the evaporator was regulated to a flow rate in therange of 120 kg/hour to 130 kg/hour at a recycle flow rate of 300kg/hour. Thus, the distillate flow rate was 123.5 kg/hour at a bottomproduct removal rate of 0.52 kg/hour. During the concentration process,a first fraction was sampled and analyzed, followed by a second fractionwhich was also analyzed. The two fractions of EGCG concentrates had atotal mass of 63.5 kg.

Table 6 shows the concentration of the tea components in the bottomproducts. The recovery of EGCG was 95.9%. The analytical result clearlyindicates that the high purity of the separated EGCG could be maintainedduring the concentration of the solution.

EGCG may be isolated from the concentrated solution in a solid formeither by spray drying or by crystallization.

TABLE 6 Concentration of the tea components in the EGCG concentrate fromthe falling film evaporator Bottom product of falling film evaporatorComposition Composition 1^(st) fraction 2^(nd) fraction Rel. Rel.Compounds HPLC/wt.-% Percentage HPLC/wt.-% Percentage Gallic acid 0.000.0 0.00 0.0 Catechin 0.17 1.5 0.14 1.6 Caffeine 0.01 0.1 0.02 0.2 EGCG10.70 95.4 8.07 94.8 Epicatechin 0.16 1.4 0.11 1.3 GCG 0.13 1.2 0.13 1.5ECG 0.05 0.4 0.04 0.5 Sum 11.22 100 8.51 100 Total mass of 39.0 kg 24.5kg solution

Example 4

450 ml of AMBERCHROM® CG-71c having a mean particle diameter of 120microns were filled into a laboratory chromatography column made fromstainless steel having an inner diameter of 2.2 cm and a length of 103cm. The column was equipped with a heating jacket. The resin was washedand equilibrated with a mixture of water/ethanol (ratio 9:1 by volume)

20 g of a concentrated catechin powder “Green tea extract, min. 95% ofpolyphenols” from Guizhou Highyin Biological Products Co. (startingmaterial) were dissolved in 20 ml of a mixture of water/ethanol (ratio9:1 by volume). Afterwards, 14 g of this solution (corresponding to 2.99g of EGCG) were applied to the top of the column. EGCG was eluted bymeans of a chromatographic pump under a pressure of 2-3 bar at atemperature of 60° C. with a mixture of water/ethanol (ratio 9:1 byvolume) under a constant flow rate of 16 ml/minute. The eluent wasdegassed and maintained under a nitrogen atmosphere prior to use. Afteran initial eluate of 2.48 l (prefraction), the flow rate was changed to25.5 ml/minute and the main eluate of 5.40 l was collected containingEGCG in a concentration of 0.627 g/l. With respect to other catechinsand caffeine, the purity of the EGCG in the main eluate determined byHPLC, and expressed as relative percentage was 97.13%. During theexperiment, the pressure in the system varied from 2 to 3 bar dependingon the flow rate applied.

Table 7 compares the concentration of the tea components in the eluateand in the starting material, thus illustrating the separation effect asshown by the relative percentage of EGCG. The concentration of the teacomponents in the starting material and in the main fraction wasdetermined by HPLC using UV absorbance and expressed as wt.-% or ppm.

TABLE 7 Separation on AMBERCHROM ® CG-71c, 60° C., solvent system:water/ethanol Tea Concentrate (starting material) Main Fraction Example4 Example 4 HPLC/ Rel. HPLC/ Rel. Compound wt.-% Percentage/% ppmPercentage/% Gallic acid 0.08 0.1 0 0.0 Catechin 0.50 0.6 1 0.2 Caffeine9.29 11.3 5 1.2 EGCG 42.23 51.4 407 97.1 Epicatechin 4.24 5.2 3 0.7 GCG8.09 9.9 1 0.2 ECG 17.70 21.6 2 0.5 Total 82.13 100 419 100

Example 5

450 ml of AMBERLITE® XAD-7, having a particle diameter of 0.3 to 1.2 mmwas packed into a laboratory chromatography column made of glass havingan inner diameter of 2.4 cm and a length of 100 cm. The column wasequipped with a heating jacket and on the bottom with a glass sinterfrit P3. The resin was thoroughly washed with deionized water andequilibrated with a mixture of water/ethanol (ratio 9:1 by volume) priorto use.

20 g of a concentrated catechin powder “Green tea extract, min. 95% ofpolyphenols” from Guizhou Highyin Biological Products Co. (startingmaterial) were dissolved in 20 ml of a mixture of water/ethanol (ratio9:1 by volume). Afterwards, 14 g of this solution (corresponding to 2.91g of EGCG) were applied to the top of the column. EGCG was eluted with amixture of water/ethanol (ratio 9:1 by volume) with a constant flow rateof 16.9 ml/minute at a temperature of 60° C. and a pressure of 0.5 to 1bar. The eluent was degassed and maintained in a nitrogen atmosphereprior to use. After an initial eluate of 2.48 l (prefraction), the flowrate was changed to 23.6 ml/minute and the main eluate of 4.95 l wascollected containing EGCG in a concentration of 0.470 g/l. With respectto other main catechins and caffeine, the purity of the EGCG in the mainfraction determined by HPLC was 86.22% (see Table 8). The yield based onEGCG was 79.8%. During the experiment, the pressure in the system variedfrom 0.5 to 1 bar depending on the flow rate applied.

To regenerate the resin, the remaining catechins were desorbed byeluting with 1.35 l of a mixture of water/ethanol (ratio 4:6 by volume)at a flow rate of 22.5 ml/minute. This fraction can also be used forfurther purification or separation of the desorbed catechins. Table 8compares the concentration of the tea components in the eluate and inthe starting material, thus illustrating the separation effect as shownby the relative percentage of EGCG. The concentration of the teacomponents in the starting material and in the main fraction wasdetermined by HPLC using UV absorbance and expressed as wt % or ppm.

TABLE 8 Separation on AMBERLITE ® XAD-7, 60° C., solvent system:water/ethanol Tea Concentrate (starting material) Main Fraction Example5 Example 5 HPLC/ Rel. HPLC/ Rel. Compound wt.-% Percentage/% ppmPercentage/% Gallic acid 0.09 0.1 0 0.0 Catechin 0.50 0.6 2 0.4 Caffeine9.17 11.5 7 1.3 EGCG 41.16 51.5 470 86.1 Epicatechin 4.16 5.2 5 0.9 GCG7.75 9.7 22 4.0 ECG 17.16 21.5 40 7.3 Total 79.99 100 546 100

Example 6

The regenerated resin of Example 5 was equilibrated in the laboratorycolumn described in Example 5 by pumping a mixture of water/ethanol(ratio 9:1 by volume) through the resin.

20 g of a concentrated catechin powder “Green tea extract, min. 95% ofpolyphenols” from Guizhou Highyin Biological Products Co. (startingmaterial) were dissolved in 20 ml of a mixture of water/ethanol (ratio9:1 by volume). Afterwards, 14 g of this solution (corresponding to 3.04g of EGCG) were applied to the top of the column. EGCG was eluted with amixture of water/ethanol (ratio 9:1 by volume) with a constant flow rateof 22.5 ml/minute at a column temperature of 40° C. and a pressure of 1to 2 bar. The eluent was degassed and maintained under a nitrogenatmosphere prior to use. After an initial eluate of 3.60 l (prefraction)the flow rate was changed to 26.3 ml/minute and the main eluate of 4.73l was collected. The EGCG concentration in the main eluate was 0.278g/l. With respect to other main catechins and caffeine, the purity ofthe epigallocatechin gallate in the main eluate determined by HPLC was93.2%. The yield based on EGCG was 42.8%. During the experiment, thepressure in the system varied from 1 to 2 bar depending on the flow rateapplied.

To regenerate the resin, the remaining catechins were desorbed byeluting with 1.98 l of a mixture of water/ethanol (ratio 4:6 by volume)at a flow rate of 26.3 ml/minute and a temperature of 40° C. Thisfraction may also be used for further purification or separation of thedesorbed catechins. Table 9 compares the concentration of the teacomponents in the eluate and in the starting material, thus illustratingthe separation effect as shown by the relative percentage of EGCG. Theconcentration of the tea components in the starting material and in themain fraction was determined by HPLC using UV absorbance and expressedas wt.-% or ppm.

TABLE 9 Separation on AMBERLITE ® XAD-7, 40° C., solvent system:water/ethanol Tea Concentrate (starting material) Main Fraction ofExample 6 Example 6 HPLC/ Rel. HPLC/ Rel. Compound wt.-% Percentage/%ppm Percentage/% Gallic acid 0.08 0.1 0 0.0 Catechin 0.51 0.6 3 1.0Caffeine 9.48 11.3 4 1.4 EGCG 43.01 51.4 276 93.2 Epicatechin 4.34 5.2 93.0 GCG 8.23 9.8 2 0.7 ECG 18.03 21.5 2 0.7 Total 83.68 100 296 100

Example 7

The regenerated resin of Example 6 was equilibrated with a mixture ofwater/isopropanol (ratio 9:1 by volume)

20 g of a concentrated catechin powder “Green tea extract, min. 95% ofpolyphenols” from Guizhou Highyin Biological Products Co. (startingmaterial) were dissolved in 20 ml of a mixture of water/isopropanol(ratio 9:1 by volume) by volume. Afterwards, 14 g of this solution(corresponding to 3.21 g of EGCG) were applied to the top of the column,and eluted with a mixture of water/isopropanol (ratio 9:1 by volume)with a constant flow rate of 18 ml/minute at a column temperature of 60°C. The eluent was degassed and maintained under a nitrogen atmosphereprior to use. After an initial eluate of 1.35 l (prefraction), the flowrate was changed to 16.5 ml/minute and a main eluate of 2.03 l wascollected. The EGCG concentration in the main eluate was 0.998 g/l. Withrespect to other main catechins and caffeine, the purity of theepigallocatechin gallate in the main eluate determined by HPLC was85.7%. The yield based on EGCG was 62.8%. During the experiment, thepressure in the system varied from 1 to 2 bar depending on the flow rateapplied.

To regenerate the resin, the remaining catechins were desorbed byeluting with 2.03 l of a mixture of water/isopropanol (ratio 4:6 byvolume) at a flow of 16.5 ml/minute and a temperature of 40° C. Thisfraction may also be used for further purification or separation of thedesorbed catechins. Table 10 compares the concentration of the teacomponents in the eluate and in the starting material, thus illustratingthe separation effect as shown by the relative percentage of EGCG. Theconcentration of the tea components in the starting material and in themain fraction was determined by HPLC using UV absorbance and expressedas wt.-% or ppm.

TABLE 10 Separation on AMBERLITE ® XAD-7, 60° C., Solvent system:water/isopropanol Tea Concentrate (starting material) Main Fraction ofExample 7 Example 7 HPLC/ Rel. HPLC/ Rel. Compound wt.-% Percentage/%ppm Percentage/% Gallic acid 0.05 0.1 0 0.0 Catechin 0.38 0.4 8 0.7Caffeine 9.48 10.8 28 2.4 EGCG 45.42 51.8 998 85.7 Epicatechin 4.38 5.016 1.4 GCG 8.80 10.0 35 3.0 ECG 19.12 21.8 80 6.9 Total 87.63 100 1165100

Example 8

The regenerated resin of Example 7 was equilibrated with a mixture ofwater/isopropanol (ratio 9:1 by volume).

20 g of a concentrated catechin powder “Green tea extract, min. 95% ofpolyphenols” from Guizhou Highyin Biological Products Co. (startingmaterial) were dissolved in 20 ml of a mixture of water/isopropanol(ratio 9:1 by volume). Afterwards, 14 g of this solution (correspondingto 3.10 g of EGCG) were applied to the top of the column. EGCG waseluted with a mixture of water/isopropanol (ratio 9:1 by volume) with aconstant flow of 16.9 ml/minute at a column temperature of 40° C. Theeluent was degassed and maintained under a nitrogen atmosphere prior touse. After an initial eluate of 2.48 l (prefraction), the flow rate waschanged to 23.66 ml/minute and a main eluate of 4.95 l was collected.The EGCG concentration in the main eluate was 0.370 g/l. With respect toother main catechins and caffeine, the purity of the EGCG in the mainfraction determined by HPLC was 86.6%. The yield based on EGCG was59.2%. During the experiment, the pressure in the system varied from 1to 2 bar depending on the flow rate applied.

To regenerate the resin, the remaining catechins were desorbed byeluting with 2.03 l of a mixture of water/isopropanol (ratio 4:6 byvolume) at a flow rate of 16.5 ml/minute and a temperature of 40° C.This fraction can also be used for further purification or separation ofthe desorbed catechins.

Table 11 compares the concentration of the tea components in the eluateand in the starting material, thus illustrating the separation effect asshown by the relative percentage of EGCG. The concentration of the teacomponents in the starting material and in the main fraction wasdetermined by HPLC using UV absorbance and expressed as wt.-% or ppm.

TABLE 11 Separation on AMBERLITE ® XAD-7, 40° C., Solvent system:water/isopropanol Tea concentrate (starting material) Main fraction ofExample 8 of Example 8 HPLC/ Rel. HPLC/ Rel. Compound wt-% Percentage/%ppm Percentage/% Gallic acid 0.20 0.2 0 0.0 Catechin 0.49 0.6 4 0.9Caffeine 9.21 10.8 6 1.4 EGCG 43.74 51.5 370 86.4 Epicatechin 4.23 5.014 3.3 GCG 8.50 10.0 11 2.6 ECG 18.52 21.8 23 5.4 Total 84.89 100 428100

Example 9 Separation of EGCG over Polyamide 11 Using Organic Solvents

A commercially available green tea extract (“Green tea extract, min. 95%of polyphenols,” Lot #960328 from Guizhou Highyin Biological ProductCo., Guiyang, China) containing catechins and caffeine in an amount asshown in Table 12 was used as the starting material. The concentrationof the tea components in the starting material was determined by HPLCusing UV absorbance.

TABLE 12 Concentration of tea components in the starting material Teaextract Tea extract Compound HPLC/wt.-% Rel. Percentage/% Gallic acid0.01 0.0 EGC 2.02 3.0 Catechin 0.78 1.2 Caffeine 8.48 12.5 EGCG 36.8754.5 Epicatechin 4.48 6.6 GCG 4.77 7.1 ECG 10.22 15.1 Total 67.63 100

250 g of a commercially available Polyamide 11 (Cat. No. 1.07435.0100,origin Merck, Darmstadt, Germany) having a particle size of 5-40 micronswere suspended in 300 ml ethyl acetate and transferred into a columnhaving an inner diameter of 5 cm and a length of 36 cm. The column wasequipped with a heating jacket and heated to 40° C. 3 g of the startinggreen tea extract, characterized in Table 12, containing 1.11 g of pureEGCG were dissolved in 153 ml of ethyl acetate and applied to the top ofthe column. An ethyl acetate/ethanol gradient elution (500 ml ethylacetate, 1000 ml ethyl acetate/ethanol (8.5:1.5 v/v), 1000 ml ethylacetate/ethanol (7:3 v/v), 2000 ml ethyl acetate/ethanol (1:1 v/v))under a pressure of 0.3 bar afforded a main fraction of 550 ml, whichafter evaporation of the solvents gave 1.12 g of solid containing 0.87 gEGCG as the main catechin component. The EGCG concentration in the maineluate was 0.186%. The yield of separated EGCG calculated from 1.106 gEGCG present in the starting tea extract was 76%.

To regenerate the resin, elution with 500 ml ethanol desorbed theremaining catechins. Before the next separation, the column wasconditioned with 500 ml of ethyl acetate.

Table 13 illustrates the separation effect. The concentration of the teacomponents in the main eluate was determined by HPLC using UVabsorbance.

TABLE 13 Concentration of tea components in the residue of the maineluate (after solvent evaporation) Residue of main Residue of mainfraction fraction Compound HPLC/wt.-% Rel. Percentage/% Gallic acid 0 0EGC 0 0 Catechin 0 0 Caffeine 0 0 EGCG 77.40 96.4 Epicatechin 0.05 0.1GCG 0.74 0.9 ECG 2.07 2.6 Total 80.26 100

Example 10 Separation of EGCG over Polyamide 11 Using an Aqueous SolventMixture

An aqueous green tea extract solution containing catechins and caffeinein an amount as shown in Table 14 was used as the starting material. Theconcentration of the tea components was determined by HPLC using UVabsorbance and expressed in wt.-%

TABLE 14 Concentration of the tea components in the residue of thestarting tea extract solution (after solvent evaporation) Tea extractTea extract Compound HPLC/wt.-% Rel. Percentage/% Gallic acid 1.36 4.8EGC 3.61 12.6 Catechin 1.45 5.1 Caffeine 6.89 24.1 EGCG 10.14 35.5Epicatechin 1.59 5.6 GCG 0.99 3.5 ECG 2.51 8.8 Total 28.54 100

25 g of Polyamide 11 (Cat. No. 1.07435.0100, origin Merck, Darmstadt,Germany) having a particle size of 5-40 microns were suspended in 100 mlwater, and the pH adjusted to 6.5. This suspension was transferred intoa column having an inner diameter of 3 cm and a length of 8 cm. 10 ml ofthe above green tea extract (Table 14) containing 0.078 g of pure EGCGwere applied to the top of the column. A water/ethanol gradient elution(500 ml water, 600 ml water/ethanol (7:3 v/v), 350 ml water/ethanol (6:4v/v), 500 ml water/ethanol (1:1 v/v)) with a flow rate of 5 ml/minuteafforded a main fraction of 110 ml (0.072 g) containing 0.046 g EGCG.The EGCG concentration in the main eluate was 0.06%. The yield ofseparated EGCG starting from 0.078 g EGCG in the tea extract was 59%.

To regenerate the resin, elution with 500 ml ethanol desorbed theremaining catechins. Before the next separation, the column wasconditioned with 500 ml of water.

Table 15 illustrates the separation effect. The concentration of the teacomponents in the main eluate was determined by HPLC using UVabsorbance.

TABLE 15 Concentration of the tea components in the residue of the mainfraction (after solvent evaporation) Residue of main Residue of mainfraction Fraction Compound HPLC/wt.-% Rel. Percentage/% Gallic acid 1.101.6 EGC 0.00 0.0 Catechin 1.29 1.9 Caffeine 0.00 0.0 EGCG 63.53 91.7Epicatechin 0.00 0.0 GCG 0.16 0.2 ECG 3.20 4.6 Total 69.28 100

Example 11 Separation of EGCG over AMBERLITE® XAD-7, Solvent System:Water/Ethanol

416 ml AMBERLITE® XAD-7 resin having a mean particle diameter between0.3 and 1.2 mm were filled into a laboratory chromatography column madefrom glass (ECO 25/999 M3V-K from Stagroma AG, Wallisellen, Switzerland)having an inner diameter of 2.5 cm and a length of 100 cm. The columnwas equipped with a heating jacket and heated to 60° C. The resin waswashed and equilibrated with a mixture of water/ethanol (ratio 9:1 byvolume).

A commercially available green tea extract (“Tea polyphenols TP-80” fromZhejang Zhongke Plant Technical Co. Ltd., Hangzhou, Zhejang, P.R. China)containing catechins and caffeine in an amount as shown in Table 16 wasused as the starting material.

The concentration of the tea components in the starting material wasdetermined by HPLC using UV absorbance and expressed in wt.-%.

TABLE 16 Concentration of tea components in the starting material Teaextract Tea extract Compound HPLC/wt.-% Rel. Percentage/% Gallic acid0.1 0.1 EGC 8.6 10.1 Catechin 1.9 2.2 Caffeine 6.2 7.3 EGCG 40.3 47.4Epicatechin 10.4 12.2 GCG 0.9 1.1 ECG 16.6 19.5 Total 85.0 100

11.2 g of the starting green tea extract, characterized in Table 16,containing 4.5 g of pure EGCG were dissolved in 112.5 ml of deionizedwater and applied to the top of the column.

The catechins were eluted with a mixture of water/ethanol (ratio 9:1 byvolume) with a constant flow rate of 0.6 l/hour at a column temperatureof 60° C. The eluent was degassed and maintained under a nitrogenatmosphere prior to use.

After an initial eluate of 1.2 l, the composition of the eluent waschanged to water/ethanol at a ratio of 8:2 by volume. This elution witha total amount of 1.5 l afforded a main fraction of 900 ml containing2.115 g EGCG. The EGCG concentration in the main fraction was 0.245%.The yield of separated EGCG starting from 4.5 g EGCG in the tea extractwas 47%. During the experiment, the pressure in the system varied from0.8 to 1.5 bar.

To regenerate the resin, the elution was continued with a mixture ofwater/ethanol 4:6 by volume, thus ethanol desorbed the remainingcatechins. Before the next separation, the column was conditioned withwater/ethanol 9:1 by volume.

Table 17 illustrates the separation effect. The concentration of the teacomponents in the residue of the main fraction (after evaporation of thesolvent) was determined by HPLC using UV absorbance and expressed aswt.-%.

TABLE 17 Concentration of tea components in the residue of the mainfraction (after solvent evaporation) Residue of main Residue of mainfraction fraction Compound HPLC/wt.-% Rel. Percentage/% Gallic acid 0 0EGC 0 0 Catechin 0.6 0.7 Caffeine 0.3 0.3 EGCG 81.4 94.5 Epicatechin 1.72.0 GCG 0.2 0.2 ECG 1.9 2.2 Total 86.1 100

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and all suchmodifications are intended to be included within the scope of thefollowing claims.

1. A process for producing (−)-epigallocatechin gallate (EGCG) comprising the steps of: a) providing a green tea extract containing tea catechins and caffeine; b) subjecting the green tea extract to chromatography on a macroporous polar resin at a temperature between about 40° C. to about 60° C.; and c) eluting the EGCG from the macroporous polar resin with a polar elution solvent at a temperature between about 40° C. to about 60° C. and at a pressure between about 0.1 bar to about 50 bar.
 2. A process according to claim 1 further comprising concentrating the eluate of step c).
 3. A process according to claim 1 further comprising regenerating the macroporous polar resin by desorbing any remaining catechins from the resin.
 4. A process according to claim 3 further comprising concentrating the desorbed catechins.
 5. A process according to claim 1 wherein the macroporous paler resin is selected from the group consisting of polyacrylate resins, polymethacrylate resins, polyamide resins, and polyester resins.
 6. A process according to claim 5 wherein the macroporous polar resin is a polyacrylate or polymethacrylate resin.
 7. A process according to claim 5 wherein the polyamide resin is Polyamide
 11. 8. A process according to claim 1 wherein steps b) and c) are carried out at a temperature between about 20° C. to about 60° C.
 9. A process according to claim 8 wherein the temperature is between about 40° C. to about 60° C.
 10. A process according to claim 1 wherein step c) is carried out under a pressure of about 0.1 bar to about 20 bar.
 11. A process according to claim 10 wherein the pressure is about 0.1 bar to about 10 bar.
 12. A process according to claim 1 wherein the polar elution solvent is a mixture of water and an organic solvent.
 13. A process according to claim 12 wherein the elution solvent is a mixture of about 70 vol % to about 95 vol % of water and about 5 vol % to about 30 vol % of an organic solvent.
 14. A process according to claim 13 wherein the elution solvent is a mixture of about 90 vol % of water and about 10 vol % at an organic solvent.
 15. A process according to claim 12 wherein the organic solvent is selected from the group consisting of ethanol, isopropanol, ethyl acetate, and acetone.
 16. A process according to claim 1 wherein the elution of step c) is carried out at a flow rate in the range of about 0.5 to about 20 bed volumes/hour.
 17. A process according to claim 16 wherein the elution is carried out at a flow rate in the range of about 0.5 to about 10 bed volumes/hour.
 18. A process according to claim 17 wherein the elution is carried out at a flow rate in the range of about 0.8 to about 5 bed volumes/hour.
 19. A process according to claim 1 further comprising concentrating an eluate from step a) by adding to the eluent an acid selected from the group consisting of citric acid, ascorbic acid, and isoascorbic acid.
 20. A process according to claim 19 wherein the amount of acid added to the eluent is about 0.1 wt % to about 2.5 wt % with respect to EGCG.
 21. A process according to claim 2 wherein the concentrating step is carried out with a pure organic solvent.
 22. A process according to claim 21 wherein the organic solvent consists of essentially about 10 vol % to about 60 vol % of water and about 40 vol % to about 90 vol % of an organic solvent.
 23. A process according to claim 22 wherein the solvent consists of essentially of a mixture of about 40 vol % of water and about 60 vol % of an organic solvent. 